Directed Mutagenesis of β-mannanase Gene from Bacillus licheniformis KD-1 for Improving Enzyme Activity and Stability

doi:10.13560/j.cnki.biotech.bull.1985.2021-0106

Abstract

Abstract:

The β-mannanase gene from Bacillus licheniformis strain KD-1 was directed mutated for improving its activity and stability. The β-mannanase gene manBl from B. licheniformis strain KD-1 was cloned and directed mutated by PCR. The gene and its mutants were expressed in B. subtilis DB104. The activities of the enzymes were measured by dinitrosalicylic acid,and DNS method. The β-mannanase ManBl and its 6 variants had the same optimal pH 6.0 and optimal temperature 60℃. All the variants had higher specific activity and pH stability between pH 6.0-10.0 than wild-type ManBl. The specific activity of the mutant ManBl（T112R/K291E）was（9 742±370.0）U/mg,which was 2.6 times of wild-type ManBl. The K_m value of ManBl（T112R/K291E）was 2.67 mg/mL. The half-life of ManBl（T112R/K291E）was 80 min at 70℃. The two enzymes with 6×His-tagged at the C terminal had lower specific activity and thermostability than the corresponding enzyme without His-tag. The β-mannanase mutant ManBl（T112R/K291E）shows potential application in feed,food and laundry industrial fields with its high specific activity,pH stability and thermostability.

Key words: β-mannanase, Bacillus licheniformis, directed mutation, enzyme activity, stability

TIAN Geng, GAO Wei-qiang, CHEN Xiao-bo, ZHANG Chun-xiao. Directed Mutagenesis of β-mannanase Gene from Bacillus licheniformis KD-1 for Improving Enzyme Activity and Stability[J]. Biotechnology Bulletin, 2021, 37(10): 100-109.

Figures/Tables 10

References 31

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质粒Plasmids	备注Notes	来源Source
pWB980		[20]
pWB-manBl	pWB980 with manBl gene	本研究This study
pWB-manBl（K291E）	pWB980 with manBl gene mutant K291E	本研究This study
pWB-manBl（T112R）	pWB980 with manBl gene mutant T112R	本研究This study
pWB-manBl（L211I）	pWB980 with manBl gene mutant L211I	本研究This study
pWB-manBl- T112R/K291E	pWB980 with manBl gene mutant T112R/K291E	本研究This study
pWB-manBl- L211I/ K291E	pWB980 with manBl gene mutant L211I/K291E	本研究This study
pWB-manBl- T112R/L211I	pWB980 with manBl gene mutant T112R/L211I	本研究This study
pWB-manBl-His	pWB980 with manBl gene fused with 6*His tagged at C terminal	本研究This study
pWB-manBl（T112R/K291E）-His	pWB980 with manBl（T112R/K291E）gene fused with 6*His tagged at C terminal	本研究This study

质粒Plasmids	备注Notes	来源Source
pWB980		[20]
pWB-manBl	pWB980 with manBl gene	本研究This study
pWB-manBl（K291E）	pWB980 with manBl gene mutant K291E	本研究This study
pWB-manBl（T112R）	pWB980 with manBl gene mutant T112R	本研究This study
pWB-manBl（L211I）	pWB980 with manBl gene mutant L211I	本研究This study
pWB-manBl- T112R/K291E	pWB980 with manBl gene mutant T112R/K291E	本研究This study
pWB-manBl- L211I/ K291E	pWB980 with manBl gene mutant L211I/K291E	本研究This study
pWB-manBl- T112R/L211I	pWB980 with manBl gene mutant T112R/L211I	本研究This study
pWB-manBl-His	pWB980 with manBl gene fused with 6*His tagged at C terminal	本研究This study
pWB-manBl（T112R/K291E）-His	pWB980 with manBl（T112R/K291E）gene fused with 6*His tagged at C terminal	本研究This study

菌株 Strain	备注Notes	来源Source
B. subtilis DB104	his,nprR2,nprE18,ΔaprA3	[21]
B. licheniformis KD-1		本研究This study
WB980	B. subtilis DB104（pWB980）,Kan^r	本研究This study
MTV13	B. subtilis DB104（pWB-manBl）,Kan^r	本研究This study
ABV3	B. subtilis DB104（pWB-manBl-K291E）,Kan^r	本研究This study
T2	B. subtilis DB104（pWB-manBl-T112R）,Kan^r	本研究This study
L4	B. subtilis DB104（pWB-manBl-L211I）,Kan^r	本研究This study
LK2	B. subtilis DB104（pWB-manBl-L211I /K291E）,Kan^r	本研究This study
TL1	B. subtilis DB104（pWB-manBl-T112R/L211I）,Kan^r	本研究This study
TK7	B. subtilis DB104（pWB-manBl-T112R/K291E）,Kan^r	本研究This study
A3	B. subtilis DB104（pWB-manBl-His6）,Kan^r	本研究This study
C5	B. subtilis DB104（pWB-manBl-T112R/L211I-His6）,Kan^r	本研究This study

菌株 Strain	备注Notes	来源Source
B. subtilis DB104	his,nprR2,nprE18,ΔaprA3	[21]
B. licheniformis KD-1		本研究This study
WB980	B. subtilis DB104（pWB980）,Kan^r	本研究This study
MTV13	B. subtilis DB104（pWB-manBl）,Kan^r	本研究This study
ABV3	B. subtilis DB104（pWB-manBl-K291E）,Kan^r	本研究This study
T2	B. subtilis DB104（pWB-manBl-T112R）,Kan^r	本研究This study
L4	B. subtilis DB104（pWB-manBl-L211I）,Kan^r	本研究This study
LK2	B. subtilis DB104（pWB-manBl-L211I /K291E）,Kan^r	本研究This study
TL1	B. subtilis DB104（pWB-manBl-T112R/L211I）,Kan^r	本研究This study
TK7	B. subtilis DB104（pWB-manBl-T112R/K291E）,Kan^r	本研究This study
A3	B. subtilis DB104（pWB-manBl-His6）,Kan^r	本研究This study
C5	B. subtilis DB104（pWB-manBl-T112R/L211I-His6）,Kan^r	本研究This study

引物名称 Primer name	引物序列Prime sequence (5'-3')
Bl man2-F1	gtgaaaaaaaRcatcgtttg
BL man2-R1	ttattccacRacaggcgtcaaag
BL man2-F2	CACACCGTTTCTCCGGTGAAC
manBT-2	GTAAGTCCCGTCTAGCCTTGCCCTTATTCCACAACAGGCGTCAAAG
amyT-F	TGTTGTGGAATAAGGGCAAGGCTAGACGGGACTTAC
amyT-R	CTAAATCGTGTCTTTCTTGGAACTTCCAGGGTATGTTTCTCTTTGATGTC
spmanB-F	AGGCGCAACTCAAGCTTTTGCCCACACCGTTTCTCCGGTGAAC
Pvf-1	ACATCAAAGAGAAACATACCCTGGAAGTTCCAAGAAAGACACGATTTAG
Pvf-2	GTTCACCGGAGAAACGGTGTGGGCAAAAGCTTGAGTTGCGCCT
R-manBl-F	GTTTCTCCGGTGAACCCGAATGCCCA
R-manBl-R	CGCCGTCCCATATCTCTCCTTTATTCA
V-manBl-F	TGAATAAAGGAGAGATATGGGACGGCG
V-manBl-R	TGGGCATTCGGGTTCACCGGAGAAAC
DR-manBl-R	GAAGTACGTCGTTTCCGGGTATTTTTCTTTG
DR-manBl-F	CAAAGAAAAATACCCGGAAACGACGTACTTC
BL-L211I-R	CGCATACACCCACAAGATATGATCCAGGCCTCT
BL-L211I-F	AGAGGCCTGGATCATATCTTGTGGGTGTATGCG
BL-T112R-R	ATAATGACCCGATCTAAACGCGGGGTTTGCGAG
BL-T112R-F	CTCGCAAACCCCGCGTTTAGATCGGGTCATTAT
V-His-F	CACCATCACCATCACCATTAAGGGCAAGGCTAGACGGGACTTAC
V-manBl-R	TGGGCATTCGGGTTCACCGGAGAAAC
R-manBl-F	GTTTCTCCGGTGAACCCGAATGCCCA
manBl-His-R	TTGCCCTTAATGGTGATGGTGATGGTGTTCCACAACAGGCGTCAAAG